We have developed a DNA nanoparticle library technology for the selection of cell binding DNA nanoparticles. Rolling circle amplification (RCA) of circular oligonucleotide templates containing randomized nucleotides produces libraries of single stranded DNA nanoparticles that can be screened for cell binding properties. The main goal of this project is to create multimodal DNA nanoparticles that specifically bind to cancer cells. The particles will be bred by a novel iterative selection and re-assortment method to create modular DNA nanoparticles that contain multiple distinct recognition elements within a single particle. This project addresses a significant challenge in many areas of cancer research and treatment, mainly the lack of cancer cell specific binding agents. Our DNA nanoparticles differ from other affinity reagents in that there is intrinsic multivalent display of the modules, allowing avidity to compensate for low monovalent affinity. The modular nature of the particle template construction allows multiple distinct recognition elements to be assembled into a single molecular entity. Furthermore, the combinatorial selection method allows the optimal particle to be evolved in the same molecular context in which it will be used. Collectively, the unique features of our DNA nanoparticle libraries represent a novel paradigm for cell affinity reagents that replaces high affinity binding to one or two defined molecular targets with a diverse landscape of high avidity interactions. The specific aims for this application are: Aim 1. Validate and optimize combinatorial selection methodology for multi- module particles. We have identified single module particles that bind to a mouse pancreatic cancer cell line. We will optimize the multi-module selection strategy with this line and confirm on two human pancreatic lines (MiaPaCa-2 and Panc-1) as well as a leukemia line (K-562) to demonstrate the feasibility against both solid and liquid tumor types. Aim 2. Demonstrate cancer specific cell binding of selected particles. Three applications will be addressed: histology, flow cytometry, and cell capture. Fluorescently labeled particles will used on tissue arrays for fluorescent microscopy and on suspension cells for flow cytometry. Particles tagged with biotin or iron oxide will used for magnetic cell separation. Aim 3. Identify the molecular targets of the cancer cell specific DNA nanoparticles. Two approaches will be used. In the first, we will perform co- precipitation experiments after crosslinking biotinylated DNA nanoparticles to the cell surface molecules. In the second, we will use click chemistry between azide or alkyne tagged particles to specifically react with cells that are either indiscriminately labeled with the partner click chemistry or with cells that contain the partner chemistry in specific protein modifications (e.g., farnesylated proteins).